Synthesis of Microbial Cyclosophoraose Derivatives Grafted Magnetic Nanoparticles

Experimental Section

Isolation of Cys. R. leguminosarum biovar viciae VF-39 was grown in 500 mL GMS medium, supplemented with 5 g mannitol and 150 mM NaCl, for 14 days at 25 °C. The carbohydrate Cys was isolated by ethanol precipitation method as previously reported,14 and purified by size chromato-graphy. The structure composition and DP were confirmed by NMR spectroscopy (Bruker 500 MHz spectrometer; AMX, Germany) and MALDI-TOF MS (Voyager-DETM STR spectro-meter; Applied Biosystems, Framingham, USA).

Synthesis of CM-Cys and CM-HP Cys. CM-Cys was prepared according to a previously reported method (Scheme 1(a)).7 We added 16.3% monochloroacetic acid solution (3.3 mL) to a mixture of Cys (200 mg) and NaOH (1.12 g) in water (3 mL). After the mixture was stirred for 4 h at 50 °C, it was neutralized with 6 N HCl, precipitated by adding 5× volume MeOH, and left overnight at 4 °C. After centrifu-gation, the precipitate was desalted using Bio-Gel P-2 (Bio-Rad Laboratories, Richmond, USA). The product was moni-tored by TLC (ethanol:buthanol:water = 5:5:4) and then identified by NMR spectroscopy and MALDI-TOF MS.

CM-HP Cys was synthesized in a two-step reaction (Scheme 1(b)). HP Cys was prepared as follows. NaOH (147.8 mg) was dissolved in 822 μL distilled water, and Cys (200 mg) was added to the solution with stirring until completely dissolved. Propylene oxide (100 μL) was added dropwise to the mixture at the freezing point with stirring. After 24 h at room temperature, the reaction was terminated by adding 5N HCl (pH = 7.0).16 A Bio-Gel P-4 column (Bio-Rad) was used to desalt the mixture, and the identity of the product was confirmed by NMR spectroscopy and MALDI-TOF MS. The carboxymethyl group was then conjugated to HP Cys, as described above, for the synthesis of CM-Cys.

Synthesis of CM-Cys- and CM-HP Cys-grafted MNPs. CM-Cys- and CM-HP Cys-grafted MNPs were synthesized using a one-step co-precipitation method (Scheme 1(c)).15 Maintaining a molar ratio of Fe2+:Fe3+ = 1:2 (17.2 mg FeCl2·4H2O and 47.2 mg FeCl3·6H2O), 83.7 mg CM-Cys / 92.6 mg CM-HP Cys were dissolved in 800 μL of distilled water with continuous stirring. After the solution was heated to 90 °C, 100 μL NH4OH (25%) was added. The reaction proceeded for 1 h at 90 °C with constant stirring in an N2 environment. The MNPs were washed with distilled water several times to remove any unreacted chemicals and were dried in a vacuum oven.

Characterization of Microbial Carbohydrate-grafted MNPs. TEM (JEOL JEM-2010; JEOL Ltd., Japan) was performed at a voltage of 80 kV to examine the morpho-logical features of MNPs. TEM samples were prepared by coating a Formvar/carbon 200-mesh copper grid with one drop of diluted particle suspension. The grid was dried at room temperature for 24 h prior to analysis.12 The nano-particle size was determined by DLS. FTIR spectroscopy (AMX) was performed in the range of 500-4000 cm−1 in a KBr matrix to characterize all MNPs. TGA was performed on a TG-DTA 2000SA thermogravimetric analyzer (Bruker), with a heating rate of 10 ℃/min from room temperature to 800 °C under N2.15